Mycologia, 105(6), 2013, pp. 1547–1554. DOI: 10.3852/13-022 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897
Penicillium kongii, a new terverticillate species isolated from plant leaves in China Bo Wang
expanding it to include nine species: P. astrolabium, P. bialowiezense, P. brevicompactum, P. buchwaldii, P. fennelliae Stolk, P. neocrassum, P. olsonii, P. spathulatum and P. tularense. In an investigation of phylloplane mycoflora in China, we found many unusual molds and thus report here on a new Penicillium species, P. kongii, belonging to section Brevicompacta.
College of Forestry, Northwest A & F University, Yangling 712100, Shaanxi, China
Long Wang1 State Key Laboratory of Mycology, the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
MATERIALS AND METHODS
Abstract: A new Penicillium species isolated from plant leaves, characterized by restricted growth, terverticillate penicilli, ovoid to ellipsoidal conidia and a red soluble pigment on yeast extract sucrose agar is reported here. Penicillium kongii sp. nov. belongs to subgenus Penicillium section Brevicompacta and is morphologically similar to P. bialowiezense and P. brevicompactum. Phylogenetic analyses based on sequence data from calmodulin gene, b-tubulin gene and rDNA ITS1-5.8SITS2 show that P. kongii forms a distinctive clade. Key words: molecular phylogeny, morphology, section Brevicompacta, taxonomy
Isolation of strains.—Leaf samples were collected from trees and kept in sterilized plastic bags. Isolation of phylloplane fungi was according to Nakase and Takashima (1993). Nineteen strains of section Brevicompacta were obtained and deposited at China General Microbiological Culture Collection (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, and the ex-type culture of P. kongii AS3.15329 was also deposited at University of Alberta Microfungus Collection and Herbarium as UAMH 11674 and USDA ARS Culture Collection as NRRL 62674. Morphological studies.—Characters were assessed with Czapek agar (CZ, Raper and Thom 1949), Czapek yeast autolysate agar (CYA, Pitt 1979), 2% malt extract agar (MEA, malt extract [Difco], Pitt 1979), YES (yeast extract sucrose agar, yeast extract (Oxoid), Frisvad and Samson 2004) and 25% glycerol nitrate agar (G25N, Pitt 1979). Color names followed Ridgway (1912). Wet mounts were prepared with material from colonies growing on CYA at 25 C after 7 d and mounted in lactophenol without dye. Microscopic examination and photography were performed with a Nikon Eclipse 80i microscope equipped with a Nikon DS-L1 digital sight system.
INTRODUCTION Species of Penicillium Link with restricted growth and complex and compact penicilli were accommodated in the P. brevicompactum series by Raper and Thom (1949). The series consisted of three members: P. brevicompactum, P. stoloniferum Thom and P. paxilli Bainier in which P. bialowiezense and P. biourgeianum Zaleski were regarded as synonyms of P. brevicompactum and P. stoloniferum respectively. Pitt (1979) relegated P. stoloniferum as a synonym of P. brevicompactum and included six species in his series Urticicola. Based on three genetic markers, Peterson (2004) revived P. biourgeianum and confirmed it was closely related to P. brevicompactum and P. olsonii. Frisvad and Samson (2004) and Scott et al. (2008) clarified that P. bialowiezense and P. biourgeianum were the same species and adopted the former as the valid name, while Frisvad and Samson (2004) included three taxa in series Olsonii, which originally had included only P. olsonii. Serra and Peterson (2007) added P. astrolabium and P. neocrassum to this series. Houbraken and Samson (2011) recognized 25 clades in Penicillium s. str. and accommodated seven members in Clade 22 (section Brevicompacta). Frisvad et al. (2013) added two new members to this section,
Molecular studies.—DNA extraction followed the method of Scott et al. (2000). Partial b-tubulin gene (BenA) sequences were amplified with primers I2: 59–CC GTC AAG ATG CGT GAG ATC GT–39 (a modified version from T1, O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995); the ITS1-5.8S-ITS2 region of the nuc rRNA gene was amplified with primers ITS5 and ITS4 (White et al. 1990); and the calmodulin gene (CaM) was amplified with primers cmdAD2 or cmdAD3 and cmdQ1 (Wang 2012). Polymerase chain reactions (PCR) were carried out in 20 mL reaction mixture containing 0.5 mL of each primer (10 pmol/mL), 1.0 mL genomic DNA (10 ng/mL), 8 mL 23 PCR MasterMix buffer (0.05 u/mL Taq polymerase, 4 mM MgCl2, 0.4 mM dNTPs) and 10 mL ultrapure sterile water (Biomed Co. Ltd, Beijng). Amplifications were performed in a PTC-150 thermo-cycler (MJ Research, Watertown, Massachusetts), which was programmed for denaturation at 94 C for 3 min, followed by 34 cycles at 94 C for 30 s, 50 C for 30 s and 72 C for 45 s, with a final elongation step at 72 C for 5 min. After amplification the PCR fragments were electrophoresed in 2.0% agarose gels with a 100 bp DNA ladder (MBI Fermentas) at 80V for 20 min.
Submitted 14 Jan 2013; accepted for publication 19 Apr 2013. 1 Corresponding author. E-mail:
[email protected]
1547
1548
MYCOLOGIA
TABLE I.
Strains included in molecular phylogenetic analyses and the GenBank accession numbers for three genetic markers Genetic markersb
Species
Strainsa
Penicillium astrolabium Serra & Peterson P. bialowiezense Zaleski
NRRL 35611T
Wine grapes, Portugal
DQ645808 DQ645793 DQ645804
NRRL 865T NRRL 2013 NRRL 32205 NRRL 32207 AS3.15314
Forest soil, Poland Mushroom spawn, Nuremburg, Germany Fresh coconut, Illinois, USA Christmas fern, Washington, USA Leaves of Berberis sp., Heihe Forest Park, Shaanxi, China Leaves of Rhododendron sp., Nangongshan Forest Park, Shaanxi, China Plant leaves, Tiantaishan Forest Park, Shaanxi, China Leaves of Rhododendron sp., Nangongshan Forest Park, Shaanxi, China Leaves of Rhododendron sp., Nangongshan Forest Park, Shaanxi, China Leaves of Cerasus sp., Shennongjia Nature Reserve, Hubei, China Gongbujiangda, Linzhi, Tibet, China Leaves of Rhododendron sp., Nangongshan Forest Park, Shaanxi, China Leaves of Rhododendron sp., Nangongshan Forest Park, Shaanxi, China Unknown Decaying mushrooms, Connecticut, USA Paprika, locality unknown Wood decay fungus, USA Wood decay fungus, Illinois, USA Leaves of Betula utilis, Heihe Forest Park, Shaanxi, China Hordeum vulgare (barley), Denmark Wheat, UK Yoghurt, Denmark Wheat, UK Leaves of Cotoneaster sp., Gongbujiangda, Linzhi, Tibet, China Plant leaves, Tiantaishan Forest Park, Shaanxi, China Plant leaves, Shennongjia Nature Reserve, Hubei, China Leaves of Cerasus sp., Shennongjia Nature Reserve, Hubei, China Leaves of Lindera obtusiloba, Heihe Forest Park, Shaanxi, China Leaves of Cotoneaster sp., Gongbujiangda, Linzhi, Tibet, China
AY484828 AY484829 AY484836 AY484837 KC427136
DQ645783 DQ645786 DQ645791 DQ645792 KC427156
AY484911 AY484897 AY484904 AY484905 KC427176
KC427137
KC427157
KC427177
KC427138
KC427158
KC427178
KC427139
KC427159
KC427179
KC427140
KC427160
KC427180
KC427141
KC427161
KC427181
KC427142 KC427143
KC427162 KC427163
KC427182 KC427183
KC427144
KC427164
KC427184
AY484817 AY484811
DQ645784 AY484912 DQ645800 AY484922
AY484818
DQ645785 AY484913
AY484824
DQ645789 AY484916
AY484825 KC427145
DQ645795 AY484917 KC427165 KC427185
JX313148
JX313182
JX313164
JX313138 JX313139 JX313147 KC427151
JX313172 JX313173 JX313181 KC427171
JX313154 JX313155 JX313163 KC427191
KC427146
KC427166
KC427186
KC427147
KC427167
KC427187
KC427148
KC427168
KC427188
KC427149
KC427169
KC427189
KC427154
KC427174
KC427194
AS3.15315
AS3.15316 AS3.15317
AS 3.15318
AS3.15319 AS3.15320 AS3.15321 AS3.15322 P. brevicompactum Diercks
NRRL 2011T NRRL 859 NRRL 2012 NRRL 28120 NRRL 28139 AS3.15323
P. buchwaldii Frisvad & Samson
P. kongii L. Wang
CBS 117181T CBS 116931 CBS 116934 CBS 116980 AS3.15329T AS3.15324 AS3.15325 AS3.15326 AS3.15327 AS3.15332
Source
CaM
BenA
ITS
WANG AND WANG: A TABLE I.
NEW TERVERTICILLATE
PENICILLIUM
1549
SPECIES
Continued Genetic markersb
Species P. neocrassum Serra & Peterson P. olsonii Bainier & Sartory
P. spathulatum Frisvad & Samson
Strainsa
Source T
CaM
BenA
ITS
NRRL 35639
Wine grapes, Madeira Island, Portugal
DQ645809 DQ645794 DQ645805
NRRL 13058T NRRL 5916 NRRL 6446 NRRL 31467 NRRL 35612 CBS 117192T CBS 116973 CBS 116974 CBS 116975 CBS 116976 CBS 116977 AS3.15328
Roots of Picea, Austria Unknown Soil, Moscow, Russia Coffee berry borer, Chiapas, Mexico Wine grapes, Portugal Moldy chestnut (Castanea sp.), France Soil under Nothofagus sp., Chile Soil under Pinus sp., Chile Soil under Raphia palm, Costa Rica Chalky soil, Denmark Chalky soil, Denmark Leaves of Cotoneaster sp., Gongbujiangda, Linzhi, Tibet, China Leaves of Cotoneaster sp., Gongbujiangda, Linzhi, Tibet, China Leaves of Cotoneaster sp., Gongbujiangda, Linzhi, Tibet, China Soil, California, USA; 5FRR 899T Cheek pouch of kangaroo rat, USA Soil, Dunhua, Jilin, China
DQ658165 AY484843 AY484844 AY484848 DQ645807 JX313149 JX313142 JX313143 JX313144 JX313145 JX313146 KC427150
DQ645797 DQ645788 DQ645796 DQ645798 DQ645801 JX313183 JX313176 JX313177 JX313178 JX313179 JX313180 KC427170
AF454076 AY484937 AY484938 AY484931 DQ645803 JX313165 JX313158 JX313159 JX313160 JX313161 JX313162 KC427190
KC427152
KC427172
KC427192
KC427153
KC427173
KC427193
AS3.15330 AS3.15331 P. tularense Paden AS3.14006T P. canescens Sopp NRRL 35656 P. saturniforme AS3.6886T (L. Wang & W.Y. Zhuang) Houbraken & Samson
KC427155 KC427175 KC427195 DQ658167 DQ658166 DQ658168 EU644062 EU644080 EU644081
a AS, China General Microbiological Culture Collection, Academia Sinica, Beijing; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; NRRL, Agricultural Research Service Culture Collection, Illinois; ex-type strains are indicated with T. b Sequences KC427136–KC427195 obtained in present study.
Gels were stained in 0.5 mg/mL ethidium bromide water solution for 15 min and examined under 254 nm UV with a portable UV light. Samples showing one single, obvious band of the anticipated length on the gel were purified and sequenced in double directions with an ABI 3700 DNA analyzer (Tsingke Biotechnologies Co. Ltd., Beijing). Raw sequences were proofread and edited manually with BioEdit 7.0.9 (Hall 1999). Edited sequences were aligned with MUSCLE of MEGA 5 (Tamura et al. 2011). Forty-eight strains from section Brevicompacta (TABLE I) were analyzed with neighbor-joining (NJ) method with Kimura 2-parameter model to calculate sequence divergence and subjected to 1000 bootstrap replications; gaps were treated as complete deletions. The three aligned datasets were submitted to TreeBASE (http:// purl.org/phylo/treebase/phylows/study/TB2:S13804).
RESULTS PCR amplification of CaM, BenA and ITS1-5.8S-ITS2 regions yielded ca. 630, 660 and 560 bp amplicons respectively. The primers for CaM generated the sequence from the second nucleotide of the codon for the ninth amino acid glutamine (Q) to the third nucleotide of the codon for the 140th amino acid
asparagine (N). The primers for BenA generated the sequence from the third nucleotide of the first intron to the third nucleotide of the 101st amino acid tryptophan (W). The trimmed alignments of CaM, BenA and ITS1-5.8S-ITS2 sequences were respectively 539, 409 and 499 characters with gaps. The phylograms resulting from the three gene matrices showed that all isolates of the new species were in a clade with P. bialowiezense, P. brevicompactum and P. neocrassum (FIG. 1; SUPPLEMENTARY FIGS. 1, 2). Although the ITS15.8S-ITS2 region showed that the six isolates of the new species clustered in one clade with a relatively low bootstrap value of 59%, the phylogenetic trees generated by CaM and BenA data both supported the status of the new taxon with robust bootstrap values of 87% and 99% respectively. TAXONOMY Penicillium kongii L. Wang, sp. nov. FIGS. 2, 3. MycoBank MB803185 Colonies 30–35 mm diam on YES at 25 C after 7 d, radially sulcate; conidiogenesis abundant, grayish
1550
MYCOLOGIA
FIG. 1. The NJ phylogram inferred from partial CaM sequence data. The bootstrap percentages over 70% derived from 1000 replicates are indicated at the nodes. Bar 5 0.02 substitutions per nucleotide position.
olive to light grayish olive (R. Pl. XLVI); coral red (R. Pl. XIII) soluble pigment characteristically produced. Colonies 18–20 mm diam on CZ at 25 C after 7 d, deep, umbonate in centers, velutinous; conidiogenesis abundant, near grayish olive to Andover green (R. Pl. XLVI, XLVII); mycelium white at margins; exudate absent or limited, brown; no soluble pigment; reverse wood brown (R. Pl. XL). Colonies 22–
26 mm diam on CYA at 25 C after 7 d, slightly deep, radially sulcate with light annular plicates, slightly convoluted in centers, margins slightly lobate, velutinous; conidiogenesis abundant, near grayish olive to Andover green (R. Pl. XLVI, XLVII); mycelium white; exudate limited to moderate, tawny to yellow ocher (R. Pl. XV); soluble pigment absent; reverse dark olive-buff to wood brown (R. Pl. XL).
WANG AND WANG: A
NEW TERVERTICILLATE
PENICILLIUM
SPECIES
1551
FIG. 2. Colonies of P. kongii AS3.15329 incubated 7 d at 25 C. A. CZ. B. CYA. C. MEA. D. YES.
Colonies 16–20 mm diam on MEA at 25 C after 7 d, low, plane, margins slightly fimbriate, velutinous; conidiogenesis abundant, near grayish olive to light grayish olive (R. Pl. XLVI); no exudate and soluble pigment; reverse uncolored. Colonies 14–18 mm diam on G25N at 25 C after 7 d, convex, plicate, velutinous with centers sparsely floccose; conidiogenesis abundant, near Andover green to pea green (R. Pl. XLVII); no exudate and soluble pigment; reverse light green-
yellow to pale green-yellow (R. Pl. V). No growth at 37 C and colonies 3–6 mm diam on CYA at 5 C after 7 d. Conidiophores arising from agar surface; stipes (100–)150–250(–400) 3 (3.5–)5–6 mm, smooth-walled; penicilli predominantly terverticillate, occasionally quarterverticillate; rami 2–4 per stipe, appressed, (7–) 10–15(–20) 3 3.5–5 mm; metulae 4–6 per ramus, 7–10 (–15) 3 3–5(–5.5) mm, clavate to apically vesiculate up to 6–9 mm diam; phialides 6–8 per metula, ampulliform
1552
MYCOLOGIA DISCUSSION
FIG. 3. P. kongii AS3.15329. A–D. Penicilli. E. Conidia. Bar 5 10 mm.
with distinguishable collula, 6–10 3 (2–)2.5–3.5 mm; conidia ovoid to ellipsoidal, 3.5–4.5 3 2–3 mm, walls smooth to finely roughened, born in short chains 120 mm long that form loose masses. Etymology: named after Prof Hua-Zhong Kong, who made great contribution to Aspergillus and Penicillium taxonomy in China. Specimens examined: CHINA. Tibet: Linzhi, Gongbujiangda, 29u549430N 93u109430E, 3400 m; ex type culture AS3.15329 from leaf sample No. GBJD3F from Cotoneaster sp., 12 Aug 2012, P-J. Han. (HOLOTYPE: HMAS 244382, the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; dried culture of ex type AS3.15329 on Czapek agar). Shaanxi: Tiantaishan Forest Park, 33u169450N, 107u039280E, 1500 m; AS3.15324 from leaf sample No. TTS14D from an unidentified plant, 4 Oct 2012, P.-J. Han. Shaanxi: Heihe Forest Park, 33u529540N, 107u479510E, 1200 m; AS3.15327 from leaf sample No. ZHH20B from Lindera obtusiloba, 28 Sep 2012, P.-J. Han. Hubei: Shennongjia Nature Reserve, 31u459350N 110u389570E, 1700 m; AS3.15325 from leaf sample No. XXY6 from an unidentified plant and AS3.15326 from leaf sample no. XXY1 from Cerasus sp., 8 Oct 2012, P.-J. Han. Tibet: Linzhi, Gongbujiangda, 29u549430N 93u109430E, 3400 m; AS3.15332 from leaf sample No. GBJD3F from Cotoneaster sp., 12 Aug 2012, P-J. Han.
Comments: Penicillium kongii is characterized by its terverticillate penicilli, ovoid to ellipsoidal conidia with smooth to finely roughened walls, and the coral red soluble pigment on YES.
P. kongii belongs to subgen. Penicillium section Cronata ser. Olsonii, based on Frisvad and Samson (2004), but a more recent phylogenetic study by Houbraken and Samson (2011) places P. kongii in Penicillium section Brevicompacta. The phylograms inferred from the three genetic markers (FIG. 1; SUPPLEMENTARY FIGS. 1, 2) show that P. kongii has some kinship with P. bialowiezense, P. brevicompactum and P. neocrassum. The designation of P. kongii as a new species was supported by both CaM and BenA loci. In the phylogram inferred from ITS (SUPPLEMENTARY FIG. 2), P. kongii fell in the clade with ‘‘P. brevicompactum’’ NRRL 2012, but the bootstrap value was not significant (59%). Penicillium kongii can be discriminated from the above species in many morphological aspects such as growth rate, soluble pigment production, and dimensions of conidiophore elements and conidia (TABLE II). In addition to the abundant red soluble pigment on YES produced by P. kongii, the striking difference between the new species and P. neocrassum is that the latter produces brown sclerotia on CYA while no sclerotia were observed in P. kongii. Moreover, P. neocrassum bears long stipes (500–800 mm) with relatively small, ellipsoidal conidia (2.5–3.5 3 2– 2.5 mm), whereas the stipes of the new species are much shorter (less than 400 mm) and conidia are ovoid to ellipsoidal and larger (3.5–4.5 3 2–3 mm) (TABLE II). Penicillium kongii is similar morphologically to P. bialowiezense and P. brevicompactum. However conidial color of P. kongii on CYA is grayish olive to Andover green, whereas that of P. bialowiezense is dark greenish glaucous to deep dull yellow green (TABLE II). Penicillium kongii also produces tawny exudate on CYA and characteristically produces soluble red pigment on YES, whereas P. bialowiezense excretes clear exudate and does not produce soluble pigment on YES. Furthermore, the conidia of P. bialowiezense differ in being globose to ellipsoidal and 2.5–3.532–3 mm. Penicillium brevicompactum is a species with high genetic diversity, as shown by CaM and BenA loci in the current work and the study of Scott et al. (2008). Pitt (1979) listed 10 synonyms for P. brevicompactum, but only P. bialowiezense has been revived (Frisvad and Samson 2004, Peterson 2004, Serra and Peterson 2007). However Scott et al. (2008) suggested that P. brevicompactum might consist of three distinct lineages. Typical isolates of P. brevicompactum grow slowly and produce robust stipes longer than 300 mm, compact terverticillate penicilli, finely roughened ellipsoidal conidia about 2.5–4 3 2–2.5 mm, and fairly
a
N/A
Fairly long tangled chains up to 180
These data integrated from our observations, Raper and Thom (1949), Pitt (1979), Peterson (2004), Frisvad and Samson (2004), and Serra and Peterson (2007).
Conidial chains (mm)
11–18 3 4–5 9–12 3 3.5–4 7–10 3 2.2–3 Ellipsoidal, 2.5–3.5 3 2–2.5
15–20 (–30) 3 4–5 9–12 (–15) 3 3.5–5 6–9 3 2.5–3 Ellipsoidal, 2.5–4 3 2–2.5
15–25 3 4–5 10–15 3 3–4.5 6–9 3 2.2–3 Globose to ellipsoidal, 2.5–3.5 3 2–3 Tangled chains up to 120
PENICILLIUM
Rami (mm) Metulae (mm) Phialides (mm) Conidia (mm)
500–800 3 4.5–6
None
N/A
15–17 Moderate, gnaphalium green to tea green
Absent Dusky drab to blackish brown
Abundant, brown Limited, clear
23–26 Sparse, gnaphalium green to tea green
N/A
P. neocrassum
300–800 3 4–6
None
20–36
12–20 Moderate to heavy, dark dull yellow-green to dark terre verte
Light brown to reddish brown Yellowish to reddish brown
None Absent or limited, yellow or reddish brown
14–20 (–25) Abundant, jade green to light cress green
9–18
P. brevicompactum
250–400 3 (3–) 4–6
None
Coral red (100–) 200–300 (–400) 3 (3.5–) 5–6 (7–) 10–15 (–20) 3 3.5–5 7–10 (–15) 3 3–5 (–5.4) 6–10 3 (2.0–) 2.5–3.5 Ovoid to ellipsoidal, 3.5–4.5 3 2–3 Tangled chains in loose masses up to 120
29–32
16–18 Abundant, deep dull yellow -green
Absent Cream
24–29 Abundant, dark greenish glaucous to deep dull yellowgreen shade None Abundant, clear, in small droplets
16–21
30–35
16–20 Abundant, grayish olive to light grayish olive
None Limited to moderate, tawny to yellow ocher, in obvious drops Absent Dark olive–buff to wood brown
22–26 Abundant, grayish olive to andover green
18–20
P. bialowiezense
NEW TERVERTICILLATE
Conidiophores Stipes (mm)
On YES at 25 C, 7 d Diam (mm) Soluble pigment
On MEA at 25 C, 7 d Diam (mm) Conidiogenesis
Soluble pigment Reverse
Sclerotia Exudate
On CYA at 25 C, 7 d Diam (mm) Conidiogenesis
On CZ at 25 C, 7 d Diam (mm)
P. kongii
TABLE II. Comparisons of morphological characters among P. kongii, P. bialowiezense, P. brevicompactum and P. neocrassuma
WANG AND WANG: A SPECIES
1553
1554
MYCOLOGIA
long, tangled conidial chains up to 180 mm; red soluble pigment is not produced on YES (TABLE II). In contrast, P. kongii grows faster than P. brevicompactum on CZ and CYA and produces relatively short stipes (100–400 mm); all six isolates in our collection excreted red soluble pigment on YES. Frisvad and Samson (2004) mentioned that a few strains of P. brevicompactum produced red pigment on YES but they did not reference individual strain numbers. It is possible that those strains represent P. kongii. ACKNOWLEDGMENTS Prof Jian-Yun Zhuang identified the plant samples. This work is supported by National Natural Science Foundation of China (NSFC No. 31270539) and National Basic Research Priorities Program of China (2012FY111600).
LITERATURE CITED Frisvad JC, Houbraken J, Popma S, Samson RA. 2013. Two new Penicillium species P. buchwaldii and P. spathulatum, producing the anticancer compound asperphenamate. FEMS Microbiol Lett 339:77–92, doi:10.1111/ 1574-6968.12054 ———, Samson RA. 2004. Polyphasic taxonomy of Penicillium subgen. Penicillium. A guide to identification of food and airborne terverticillate Penicillia and their mycotoxins. Stud Mycol 49:1–173. Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. App Environ Microbiol 61:1323–1330. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. Houbraken J, Samson RA. 2011. Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Stud Mycol 70: 1– 55, doi:10.3114/ sim.2011.70.01 Nakase T, Takashima M. 1993. A simple procedure for the high frequency isolation of new taxa of ballistosporous yeasts living on the surfaces of plants. Riken Rev 3:33–34.
O’Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116, doi:10.1006/mpev.1996.0376 Peterson SW. 2004. Multilocus DNA sequenced analysis shows that Penicillium biourgeianum is a distinct species closely related to P. brevicompactum and P. olsonii. Mycol Res 108:434–440, doi:10.1017/S0953756204009761 Pitt JI. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London: Academic Press. 632 p. Raper KB, Thom C. 1949. A manual of the penicillia. Baltimore, Maryland: Williams & Wilkins. 875 p. Ridgway R. 1912. Color standards and color nomenclature. Washington, DC: Published by the author. 43 p, 53 p. Scott J, Malloch D, Wong B, Sawa T, Straus N. 2000. DNA heteroduplex fingerprinting in Penicillium. In: Samson RA, Pitt JI, eds. Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Amsterdam, the Netherlands: Harwood Academic Publishers. p 225–236. Scott JA, Wong B, Summerbell RC, Untereiner WA. 2008. A survey of Penicillium brevicompactum and P. bialowiezense from indoor environments, with commentary on the taxonomy of the P. brevicompactum group. Botany 86:732–741, doi:10.1139/B08-060 Serra R, Peterson SW. 2007. Penicillium astrolabium and Penicillium neocrassum, two new species isolated from grapes and their phylogenetic placement in the P. brevicompactum and P. olsonii clade. Mycologia 99:78– 87, doi:10.3852/mycologia.99.1.78 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA 5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739, doi:10.1093/molbev/msr121 Wang L. 2012. Four new records of Aspergillus section Usti from Shandong Province, China. Mycotaxon 120:373– 384, doi:10.5248/120.373 White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MS, Gelfand DH, eds. PCR protocols: a guide to methods and applications. New York: Academic Press. p 315–322.
